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Abstract
Thymus is one of the primary lymphoid organs—the major source of self-restricted, self-tolerant naïve T cells required for robust adaptive immunity system. However, through aging, thymus is one of the earliest organs that start losing its function—which is also known as thymus involution. Thymus involution leads to the decrease of immune function, which significantly increases the risk of diseases, such as cancer and auto-immunity disease. Thus, finding a sufficient method to rescue the thymus function caused by thymus involution & thymus abnormalities is really significant. Although some evidence has shown that transplant neonatal thymus as well as cytokine induction could partial rescue thymus function, these methods are all highly limited by recourses and effective time period. Our lab previously proved that by over-expressing thymus master transcription factor Foxn1 in Mouse Embryonic fibroblasts (MEFs), we can successfully reprogram these MEFs into induced functional thymus epithelial cells (iTECs). These iTECs express many thymus related genes such as DLL4, CCL25 and Krt5. Also, these reprogrammed iTECs successfully support T cells development both in vitro & in vivo. However, the detailed mechanisms of this iTECs reprogramming and how similar it is compared to real fetal TECs is still being uncovered.
This dissertation presents detailed analysis of the reprogramming mechanisms during iTEC reprogramming as well as gene expression profile comparison to E14.5 fetal TECs. Specifically, I utilized bulk RNA-sequencing to re-construct gene expression profile through different time point of reprogramming. I analyzed these RNA-sequencing results and show that iTECs reprogramming process acts in a step by step manner, in which the early stage reprogramming cell population and the late stage reprogramming cell population is very distinct. I also show that many different transcription factors and pathway plays a crucial role in iTECs reprogramming, such as Notch pathway and cell cycle related pathway. Further experiment data shows that by modifying these pathways, we can make iTECs turning on important medullary transcription factor, increase the reprogramming efficiency as well as rescue the cell cycle arrest phenotype. Furthermore, I provide total gene expression profile comparison between different stages of iTECs and E14.5 fetal TECs that suggest the even the late stage iTECs is still highly different compared to fetal TECs, while several pathways may be potential target to further increase the similirty between iTECs and fetal TECs. Finally, I discuss possible ways to generate iTECs using non-transgenic crispr-dCAS9 methods that would be potential useful for future clinical application in both mouse and human cells.